通过体外分子进化技术获得耐高温β-葡萄糖苷酸酶的研究  

Molecular Evolution of Beta-Glucuronidase in vitro : Obtaining Thermotolerant GUS Gene

作　　者：熊爱生[1] 姚泉洪[1] 彭日荷[1] 陈建民[2] 李贤[1] 范惠琴[1] 

机构地区：[1]上海市农业遗传育种重点实验室上海市农业科学院生物技术研究中心,上海201106 [2]扬州大学生物科学与技术学院,扬州225009

出　　处：《Acta Genetica Sinica》2002年第11期1034-1040,共7页

基　　金：上海市农业科学院青年基金项目 (资助号 :2 0 0 1-0 9-0 1-1)~~

摘　　要：采用高保真聚合酶从质粒pBI12 1中扩增出 1.8kb的专一条带 ,克隆入pBluescriptSK载体 ,测序结果显示与报道一致。该克隆GUS基因被用作对照 ,再以此为模板 ,通过DNA重排技术 ,经DNaseⅠ降解 ,PrimerlessPCR ,PrimerPCR重新得到大量的突变GUS基因。这些突变的GUS基因构建入原核表达载体pG2 5 1中 ,电击转化大肠杆菌菌株DH5α ,构建GUS基因突变体库 ,经过 3轮的重排、筛选得到 80℃处理 30分钟仍显示较高活性的耐高温GUS基因GUS3 3,基因测序显示 ,GUS3 3与GUS基因之间的同源性为 99.2 % ,核苷酸序列推导的氨基酸序列显示 ,11个氨基酸发生了突变 ,80 %的突变集中在蛋白的C端。Tm值GUS3 3为 80℃ ,GUS ck为 5 5℃ ,提高了 2 5℃ ,GUS3 3β -葡萄糖苷酸酶热稳定性有了很大的提高 ,GUS3 3酶的最适温度明显提高。The Escherichia coli beta glucuronidase gene ( gus ) has been developed as a reporter gene for plants, and has been widely used for over a decade. Both chromogenic and fluorogenic GUS substrates have been synthesized, allowing rapid nonradioactive assays. The use of the Escherichia coli enzyme beta glucuronidase (GUS) as a reporter in gene expression studies is limited by some plants and plant associated bacteria express endogenous glucuronidase activities. The use of the enzyme as a reporter in transgenic plants is limited by high false positive. Laboratory evolution methods were used to enhance the thermostability and activity of the beta glucuronidase. Using plasmid pBI121 as template, a 1.8kb specific product was amplified and cloned into the vector pBluescript SK. The result of nucleotide sequence analysis was the same as reported. In vitro recombination (DNA shuffling), which involves DNase Ⅰ digestion, primerless PCR, and primer PCR was used togenerate mutant libraries. The mutant GUS3 3 gene was isolated after three rounds of mutation, DNA shuffling, and screening. The GUS3 3 enzyme can resistant high temperature up to 80℃ for 30min. The nucleotide sequence analysis showed 99.2% homology between the GUS ck gene from pBI121 and GUS3 3 gene. The deduced amino acid sequence demonstrated that 11 amino acid was changed. The Tm value of GUS3 3 is 80℃ and increased by 25℃ above GUS ck (55℃). The researches indicated the feasibility of the molecular evolution of beta glucuronidase in vitro to improve enzymatic thermostability.

关 键 词：体外分子进化技术 β-葡萄糖苷酸酶 基因重排 耐高温 植物 

分 类 号：Q78[生物学—分子生物学]